The expression of constitutively active JAK2 kinase in hematopoietic stem cells is sufficient to induce myeloproliferative neoplasms (MPNs) in mice paved the way for developing JAK2-targeted therapies. Recent clinical success of JAK2 inhibitors (INCB018424 and TG101348) in the treatment of MPN patients are promising, and most likely these two clinical agents will be approved by FDA for the treatment of JAK2 mediated disorders. Given the structural plasticity in kinases and emergence of resistance to kinase inhibitor therapy we anticipate that resistance to JAK2 inhibitors will develop as well. Additionally, our current understandings of JAK2 regulatory mechanisms are speculative. For instance, how somatic mutations activate the kinase is not fully understood that limits us to develop efficient therapeutic development. Mutagenesis studies coupled with in silico structural modeling provide greater structural-functional insights, patient management and drug design. Toward this end, we propose to identify the drug resistant mutations for JAK2 clinical inhibitors to understand the mechanisms of drug resistance and JAK2 regulatory mechanisms. Our preliminary data suggests that the frequency of resistance against INCB018424 is higher as compared to TG101348 suggesting their selectivity for distinct kinase conformations. We hypothesize that these inhibitors will select for different constellation of mutations and may complement each other to suppress the development of resistance. In this study, we propose to perform mutational studies coupled with biochemical and structural characterization to understand the mechanisms of drug resistance and JAK2 regulation that will help us patient diagnosis, drug design and developing strategies to combat clinical resistance.